Human cytomegalovirus (HCMV) infects over half of all Veterans and threatens the lives of those with impaired immune systems. Even among Veterans with normal immune systems, the insidious reactivation of the virus taxes the immune system, incites low-level inflammation, and possibly accelerates aging and shortens lifespan. HCMV is the most common infectious cause of birth defects. There is no HCMV vaccine and the antiviral drugs have problems with potency, toxicity, and drug-resistance. The long-range goal of this research is to identify critical pivot points in the viral transcription-DNA replication cycle that are vulnerable new targets for therapeutic intervention. This proposal is based on the premise that our gap in knowledge of how viral early transcription begets viral DNA replication and viral DNA replication begets viral late transcription limits our ability to design therapeutic treatments for the viral disease. We have developed modified PRO-Seq and PRO-Cap methods to determine exactly where on the viral genome that Pol II and its attached nascent transcript is located and at what frequency the engaged Pol II is at that nucleotide position during the infection. This allows us to precisely determine where transcription is initiating, the extent of promoter-proximal Pol II pausing, and the degree to which productive transcription elongation is taking place. We designed bioinformatics algorithms to analyze this data. Our preliminary studies show that HCMV utilizes host Pol II elongation control in early and late infection, but HCMV evolved different strategies in promoting the viral transcription and linking it to viral DNA replication. Frequently used viral promoters more often contain upstream TATA elements than do host promoters and viral initiator elements differ from that of the host in nucleotide preference. In late infection, HCMV and not the host uses TATT as a Pol II positioning element, which presumably requires the actions of viral late transcription factors (LTFs). We also find that nearly 20% of paused Pol II is found in the non-coding long RNA4.9 gene at a location in the viral lytic origin of replication (oriLyt) that is essential for oriLyt function; robust enhancer transcription underlies the world renown viral major immediate-early promoter; and viral transcription is pervasive and exhibits a pattern predictive of that arising from an unchromatinzed DNA template. Our research plan is designed to further validate, establish meaning of, and mechanistically understand these findings. To advance these objectives, we have adapted a newly developed approach to specifically and rapidly deplete viral proteins putatively involved in viral transcription. As proof of concept, we observe changes in levels of multiple viral RNAs after eliminating all the viral IE2 protein isoforms in late infection over a 6-hr timeframe, suggesting that the actions of one or more of these viral protein isoforms may have an overarching effect on viral transcription. We will combine this technique with PRO-Seq and PRO-Cap to determine the roles of the IE2 isoforms and the UL79 LTF (a putative late viral transcription elongation factor) in viral late transcription. In doing these studies, we will test the hypothesis that HCMV usurps Pol II initiation and elongation control to direct viral DNA replication, coordinate the virus?s gene expression program in lytic and latent-like infections, and contend with the threat of chromatin invasion. Our studies have been specifically designed to determine the role of transcription and structural elements in the function of the HCMV replication origin (Aim 1); determine the core DNA elements and viral factors required in late viral transcription (Aim 2); and determine how transcription differs in a quiescent infection and responds to activation (Aim 3). This proposal integrates the pertinent and extensive expertise of the Meier and the Price labs in virology and transcription, respectively. This research could not be done by either lab alone. The Meier and Price labs have a strong foundation of productive collaboration on which to build and complete this research plan.